Prohemostatic proteins for the treatment of bleeding

ABSTRACT

This disclosure relates to recombinant FXa polypeptides that can be used as antidotes to completely or partially reverse an anti-coagulant effect of a coagulation inhibitor in a subject, preferably a direct factor Xa inhibitor. Disclosed herein are recombinant factor Xa proteins and a method of completely or partially reversing an anti-coagulant effect of a coagulation inhibitor in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 15/313,881, filed Nov. 23, 2016, which claims the benefit under35 U.S.C. § 371 of International Patent Application PCT/NL2015/050377,filed May 26, 2015, designating the United States of America andpublished in English as International Patent Publication WO 2015/183085A1 on Dec. 3, 2015, which claims the benefit under Article 8 of thePatent Cooperation Treaty to European Patent Application Serial No.14169895.1, filed May 26, 2014, the contents of the entirety of each ofwhich are incorporated herein by this reference.

TECHNICAL FIELD

This disclosure is in the field of medical treatment. In particular, thedisclosure is in the field of treating, preventing or amelioratingbleeding complications resulting from a modulated hemostatic response.

BACKGROUND

Millions of patients worldwide require anticoagulant drugs for theprophylactic management of stroke in atrial fibrillation or preventionand treatment of venous thrombosis. Prophylaxis is traditionallycentered on the coumarin-based oral anticoagulant Vitamin K Antagonists(VKAs) such as Warfarin, Acenocoumarol and Phenprocoumon, which blockthe synthesis of vitamin K-dependent blood coagulation factors. Furtheranticoagulant drugs include target-specific anticoagulants, such asdabigatran, that inhibit the enzyme thrombin, which is a serine proteasethat converts soluble fibrinogen into insoluble strands of fibrin.Efficacious reversal of the anticoagulant effect, with a so-calledantidote, is a prerequisite for safe drug usage. This is particularlyimportant considering that, just in the Netherlands alone, over 10,000patients treated with anticoagulants annually suffer from an adversesevere bleeding event, including up to 2,000 fatalities (H. Adriaansen,et al., “Samenvatting Medische Jaarverslagen van de Federatie vanNederlandse Trombosediensten,” 2011; 1-44).

Currently available anticoagulant-antidote pairs to preventover-anticoagulation are heparin-protamine and warfarin-vitamin K.Prothrombin complex concentrates (PCC) containing vitamin K-dependentcoagulation factors II, IX, X (3-factor PCC) or II, VII, IX, X (4-factorPCC) and varying amounts of proteins C and S have been indicated for thereversal of warfarin-related effects (see, for example, Frumkin, Ann.Emerg. Med. 2013, 62:616-626). Fresh frozen plasma and recombinantfactor VIIa (rfVIIa) have also been used as non-specific antidotes inpatients under low molecular weight heparin treatment, suffering frommajor trauma or severe hemorrhage (Lauritzen et al., Blood 2005,106:2149, Abstract 607A-608A). Also reported are protamine fragments(U.S. Pat. No. 6,624,141) and small synthetic peptides (U.S. Pat. No.6,200,955) as heparin or low molecular weight heparin antidotes; andthrombin muteins (U.S. Pat. No. 6,060,300) as antidotes for thrombininhibitors. Prothrombin intermediates and derivatives have been reportedas antidotes to hirudin and other thrombin inhibitors (U.S. Pat. Nos.5,817,309 and 6,086,871). Despite the absence of solid clinical data,dabigatran-associated severe bleeding is preferably treated with thenon-specific reversal agent activated prothrombin complex concentrate(APCC) (Siegal et al., Blood 2014, 123:1152-1158).

Newly developed direct factor Xa (FXa) inhibitors (DFXIs), such asrivaroxaban, apixaban and edoxaban, are anticoagulants and may largelyreplace the classic VKAs in the near future because of their rapidtherapeutic effectiveness, ease of dosing and lack of monitoringrequirements due to fewer drug and food interactions and predictablepharmacokinetics. DFXIs are small compound inhibitors that have beenspecifically designed to tightly bind to and halt the activity of bloodcoagulation FXa. Coagulation FXa is an essential serine protease thatnormally circulates as an ˜60 kDa inactive precursor (zymogen)coagulation factor X (FX) in blood, but is converted upon vasculardamage to its active protease form in a complex series of proteinactivation steps, collectively known as the blood coagulation cascade.Central to this system is the formation of the cofactor-protease complexknown as the prothrombinase complex that consists of coagulation FXa inassociation with the cofactor factor Va (FVa), which assembleexclusively on a negatively charged phospholipid membrane and convertinactive prothrombin into the active serine protease thrombin.

A major drawback to the use of the DFXIs is the absence of a specificand adequate reversal strategy to prevent and stop potentiallife-threatening bleeding complications associated with itsanticoagulant therapy.

Since DFXIs inhibit both free and prothrombinase-bound coagulation FXa(European Medicines Agency, 2008, CHMP assessment report for Xarelto,Procedure No. EMEA/H/C/000944, Doc.Ref.: EMEA/543519/2008), effectiverestoration of normal hemostasis would, therefore, require either fullreplacement of circulating coagulation FXa or effective removal ofinhibitory compounds from blood.

Currently, there are no specific reversal strategies available toprevent and stop potential life-threatening bleeding complicationsassociated with DFXI therapy. Next to life-supporting and surgicaltherapies, non-specific reversal therapy using 3- and 4-factor PCC maybe considered based on limited evidence (Siegal et al., Blood 2014,123:1152-1158; Levi et al., J. Thrombosis Haemostatis 2014, Publishedonline 8 May 2014; doi: 10.1111/jth.12599). A reversal strategy specificfor DFXI-associated bleeding is in development, which is based on acatalytically inactive form of recombinant FXa (andexanet alpha) thatserves as a decoy for DFXIs by binding and thereby trapping circulatingDFXIs, thereby enabling endogenous coagulation FXa to normallyparticipate in coagulation (Lu et al., Nature Medicine 2013, 19:446). Adownside to this approach is that high doses of andexanet alpha need tobe administered since stoichiometric concentrations are required toattain inhibition (400 mg IV bolus in phase III trial; Portola NewsRelease Mar. 19, 2014). Furthermore, since the half life of DFXIpartially depends on renal clearance, the amount of decoy FXa requiredto trap all circulating inhibitory molecules may even be higher in thecase of renal failure. This reversal strategy does not provide a fastand direct procoagulant response, as the response is dependent on thegeneration of free, endogenous coagulation FXa.

At this moment, a direct, adequate reversal strategy to prevent and stoppotential life-threatening bleeding complications associated with DFXIanticoagulant therapy is not available.

BRIEF SUMMARY

This disclosure solves this problem by providing, as an adequatereversal strategy to prevent and stop potential life-threateningbleeding complications associated with DFXI anticoagulant therapy, arecombinant protein comprising, or consisting of, a mammalian,preferably primate, more preferably human, coagulation FXa polypeptide,the polypeptide having an alteration in a region of amino acid residuescorresponding to the region of amino acid residues between Gly-289 andAsp-320 of SEQ ID NO: 1, preferably between Glu-297 and Asp-320 of SEQID NO: 1, more preferably between Val-305 and Asp 320 of SEQ ID NO: 1and most preferably between His-311 and Asp-320 or between His-311 andTyr-319 of SEQ ID NO: 1, wherein the alteration is an insertion and/orreplacement and/or deletion of at least one amino acid residue,preferably an insertion of at least one amino acid residue. Forclarification purposes, the amino acid residue numbering is based on thehuman coagulation FX amino acid sequence as provided in SEQ ID NO: 1.

It was found that a catalytically active human coagulation FXa, with analtered amino acid composition at a region between the Gly and Aspcorresponding to Gly 289 and Asp-320 of SEQ ID NO: 1, participates inthe coagulation cascade as a procoagulant, whereby the factor has adecreased sensitivity to inhibition by DFXIs, compared to a coagulationFXa not having the altered amino acid composition. This disclosureprovides, therefore, a procoagulant antidote that does not depend on thegeneration of free, endogenous coagulation FXa and offers a fast anddirect reversal strategy to prevent and stop complications associatedwith DFXI anticoagulant therapy.

The amino acid sequence of human coagulation FX is provided in SEQ IDNO:1 and can be found in GENBANK® under “AAH46125.1”. The amino acidresidue numbering in this sequence is based on the human coagulation FXsequence. Coagulation FX with the sequence listed in SEQ ID NO:1 is aprecursor containing a prepro-leader sequence (amino acid residues 1 to40 of SEQ ID NO:1), followed by sequences corresponding to a coagulationFX light chain (amino acid residues 41 to 179 of SEQ ID NO:1), a RKRtriplet (amino acid residues 180 to 182 of SEQ ID NO:1) which is removedduring secretion of coagulation FX, and a coagulation FX heavy chain(amino acid residues 183 to 488 of SEQ ID NO:1) containing theactivation peptide (AP) (amino acid residues 183 to 234 of SEQ ID NO:1)and the catalytic serine protease domain (amino acid residues 235 to 488of SEQ ID NO:1).

Maturation of human coagulation FX involves inter alia proteolyticcleavage and post-translational modification in the Golgi apparatus. Themature FX protein is a two-chain molecule, composed of a light chain anda heavy chain that are linked by a disulfide bond (Uprichard et al.,Blood Reviews 2002, 16:97-110). Mature human coagulation FX is activatedby cleavage of a peptide bond on the heavy chain between Arg 234 andIle-235 of SEQ ID NO: 1, thereby releasing a 52-residue activationpeptide from the heavy chain of coagulation FX. The resultingdisulfide-linked light chain and truncated heavy chain constitute anactivated FXa polypeptide.

The amino acid sequence of the light chain of human coagulation FXa isprovided in SEQ ID NO:2. The amino acid sequence of the heavy chain ofhuman coagulation FXa is provided in SEQ ID NO:3.

The term “recombinant,” as used herein, refers to a protein that isproduced using recombinant DNA techniques known to the person skilled inthe art. A recombinant coagulation FX or FXa polypeptide is alsoindicated as rFX or rFXa. A recombinant protein preferably is notidentical to a native protein, for example, because the amino acidcomposition differs and/or because of a difference in post-translationalmodification such as glycosylation.

The term “alteration,” as used herein, refers to an insertion and/orreplacement and/or deletion of at least one amino acid residue. Thealteration preferably is an insertion of at least one amino acid.

The phrase “recombinant protein comprising a coagulation FXapolypeptide,” as used herein, is meant to encompass a protein thatcomprises a recombinant coagulation FXa polypeptide, preferablymammalian, more preferably primate, and most preferably of human origin.The phrase includes, for example, a recombinant mammalian precursorprotein, such as human coagulation FX, that is processed and/oractivated into a mammalian coagulation rFXa polypeptide. Thus, a proteinof the disclosure is preferably a recombinant mammalian, preferablyprimate, more preferably human coagulation FX, having an insertionand/or replacement and/or deletion, preferably an insertion, of at leastone amino acid residue in a region of amino acid residues correspondingto the region of amino acid residues between Gly-289 and Asp-320 of SEQID NO: 1, preferably between Glu-297 and Asp-320 of SEQ ID NO: 1, morepreferably between Val-305 and Asp-320 of SEQ ID NO: 1 and mostpreferably between His-311 and Asp-320 of SEQ ID NO:1. In addition, thephrase includes a protein that comprises one or more additional aminoacid sequences, besides the coagulation rFXa polypeptide, for example,an amino acid sequence that constitutes a tag, for example, a FLAG tagas described in EP0150126, and/or one or more other identificationpeptides.

In one embodiment, therefore, a recombinant protein comprising acoagulation FXa polypeptide according to the disclosure is a coagulationfactor X polypeptide, the polypeptide having an alteration in a regionof amino acid residues corresponding to the region of amino acidresidues between Gly-289 and Asp-320, preferably between His-311 andAsp-320 of SEQ ID NO:1; wherein the alteration is an insertion of atleast one amino acid residue.

The term “coagulation FX,” as used herein, refers to an inactivecoagulation FX precursor protein. The skilled person knows thatcoagulation FX is also referred to as preproprotein FX. As is usedherein, a coagulation FX comprises a coagulation FXa polypeptide.

The term “mature coagulation FX,” as used herein, refers to an inactivecoagulation FX protein that is composed of a light chain and a heavychain that are linked by a disulfide bond. This FX protein is alsoreferred to as proprotein FX, or zymogen FX. As is used herein, a maturecoagulation FX comprises a coagulation FXa polypeptide.

A protein of the disclosure preferably comprises, or is, a mammalian,preferably primate, more preferably human or humanized, coagulation FXapolypeptide, having an insertion and/or replacement and/or deletion,preferably an insertion, of at least one amino acid residue in a regionof amino acid residues corresponding to the region of amino acidresidues between Gly-289 and Asp-320 of SEQ ID NO:1.

The term “humanized,” as is used herein, refers to the replacement orhumanization of preferably exterior amino acid residues of a protein ofone species for amino acid residues that are present in a humanhomologue of the protein so that the proteins of the first species willnot be immunogenic, or are less immunogenic, when applied to a human.The replacement of exterior residues preferably has little, or no,effect on the interior domains, or on the interdomain contacts betweenlight and heavy chains. A protein of the disclosure of non-human origin,preferably mammalian origin, more preferably primate origin, ispreferably humanized in order to reduce the immunogenicity of theprotein in a human.

A non-human protein of the disclosure preferably comprises a humanizedmammalian, more preferably a humanized primate, coagulation FXapolypeptide, as the risk of an antigenic response upon administration inthe human body is expected to be lower as compared to a protein of thedisclosure comprising a non-humanized coagulation FXa polypeptide.

In the context of humanizing proteins, attention can be paid to theprocess of humanizing that is applicable to antibodies. This processmakes use of the available sequence data for human antibody variabledomains compiled by Kabat et al., “Sequences of Proteins ofImmunological Interest,” 4th ed. (1987), Bethesda, Md., NationalInstitutes of Health, updates to this database, and other accessibleU.S. and foreign databases (both nucleic acid and protein). Non-limitingexamples of the methods used to generate humanized antibodies include EP519596; U.S. Pat. No. 6,797,492; and described in Padlan et al., Mol.Immunol. 1991, 28:489-498. Further exemplifying the process ofhumanization of non-human proteins, Sarkar et al., “Joumal of Lipids”2012, Article ID 610937, p. 1-13, described that Paraoxonase-1 wassuccessfully humanized by altering the surface of the enzyme to reflectthe human sequence.

The term “coagulation FXa polypeptide” refers to the catalyticallyactive form of a coagulation FX. The coagulation FXa polypeptide isobtained by cleavage of the activation peptide from the heavy chain of amature coagulation FX. A coagulation FXa polypeptide activatesprothrombin and, as a result, promotes coagulation. In the context ofthe disclosure, a protein is a coagulation FXa polypeptide if it is aprocoagulant serine protease and if the full-length amino acid sequenceof the protein comprises stretches of, or single, amino acid residuesthat correspond to stretches of, or single, amino acid residues that areconserved between coagulation FX factors of different species, as isindicated in FIGS. 8A-8C. For example, a procoagulant serine proteasecomprising a polypeptide that contains stretches of amino acid residuesthat correspond to amino acid residues Cys-246 to Ala-250, Phe-260 toLeu-266 and/or Asp-413 to His-423 of SEQ ID NO:1, is assumed to be acoagulation FXa polypeptide. The coagulation FXa polypeptide ispreferably obtained by local and/or topical application of a recombinantprotein according to the disclosure. Methods to determine whether aprotein is a serine protease are known in the art and include sequencecomparison and use of a protease detection kit, for example, fromSigma-Aldrich.

The term “mammalian coagulation FXa polypeptide,” as used herein, refersto a coagulation FXa polypeptide that is endogenously present in amammal, preferably a primate, more preferably a human.

The term “coagulation inhibitor,” as used herein, refers to ananti-coagulation agent. The term “coagulation inhibitor” includes, butis not limited to: (i) agents, such as heparin, that stimulate theactivity of antithrombin, (ii) coumarin-based oral anticoagulant vitaminK antagonists, such as warfarin, acenocoumarol and phenprocoumon, and(iii) DFXIs.

The term “DFXI,” as used herein, refers to direct FXa inhibitors, forexample, oral direct FXa inhibitors. DFXIs are small compound inhibitorsthat bind to and halt the activity of coagulation FXa. The group ofDFXIs includes, but is not limited to, rivaroxaban(5-chloro-N-[[(5S)-2-oxo-3-[4-(3-oxo-4-morpholinyl)phenyl]-5-oxazolidinyl]methyl]-2-thiophenecarboxamide),apixaban(1-(4methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide),edoxaban(N′-(5-chloropyridin-2-yl)-N-[(1S,2R,4S)-4-(dimethylcarbamoyl)-2-[(5-methyl-6,7-dihydro-4H-[1,3]thiazolo[5,4-c]pyridine-2-carbonyl)amino]cyclohexyl]oxamide;4-methylbenzenesulfonic acid), betrixaban(N-(5-chloropyridin-2-yl)-2-[[4-(N,N-dimethylcarbamimidoyl)benzoyl]amino]-5-methoxybenzamide),darexaban(N-[2-[[4-(Hexahydro-4-methyl-1H-1,4-diazepin-1-yl)benzoyl]amino]-3-hydroxyphenyl]-4-methoxybenzamide),otamixaban (methyl(2R,3R)-2-[(3-carbamimidoylphenyl)methyl]-3-[[4-(1-oxidopyridin-1-ium-4-yl)benzoyl]amino]butanoate),eribaxaban(2R,4R)-1-N-(4-chlorophenyl)-2-N-[2-fluoro-4-(2-oxopyridin-1-yl)phenyl]-4-methoxypyrrolidine-1,2-dicarboxamide),letaxaban(1-[1-[(2S)-3-(6-chloronaphthalen-2-yl)sulfonyl-2-hydroxypropanoyl]piperidin-4-yl]-1,3-diazinan-2-one,LY517717(N-[2-[4-(1-methylpiperidin-4-yl)piperazin-1-yl]-2-oxo-1-phenylethyl]-1H-indole-6-carboxamide)and 813893(N-cyclohexyl-N-[2-[(4-methyl-1,3-thiazol-2-yl)amino]-2-oxoethyl]furan-2-carboxamide).The terms “DOAC” (direct oral anticoagulant) and “DFXI” are usedinterchangeably herein.

The term “homologous,” as used herein, refers to amino acid sequenceidentity between two amino acid sequences, expressed as a percentage ofthe total length of the two amino acid sequences. Sequence identity isdetermined by comparing the identity of individual amino acid residuesof an amino acid sequence to the corresponding amino acid residue inanother amino acid sequence.

The term “region,” as used herein, refers to a stretch of amino acidresidues that is bordered by two amino acid residues. The numbering ofamino acid residues as applied herein is based on the amino acidsequence of SEQ ID NO: 1.

The term “insertion” or “inserted,” as used herein, refers to theaddition of amino acid residues in a specific region of a nativecoagulation FXa polypeptide, thereby increasing the number of amino acidresidues in the region, compared to the number of amino acid residues inthat region of the native coagulation factor FXa polypeptide.

The term “replacement” or “replaced,” as used herein, refers to thesubstitution of one or more amino acid residues in a specific region, orat a specific site, of a coagulation factor Xa polypeptide, therebyaltering the amino acid sequence, but not the number of amino acidresidues in the region. A replacement is the consequence of the deletionof an amino acid residue followed by the insertion of a different aminoacid residue at the same position.

The term “deletion” or “deleted,” as used herein, refers to deleting oneor more amino acid residues in a specific region, or at a specific site,of a coagulation factor Xa polypeptide, thereby reducing the number ofamino acid residues in the region of the polypeptide.

The term “native coagulation FXa polypeptide,” as used herein, refers toan endogenous coagulation FXa polypeptide that naturally occurs in ananimal, preferably in a mammal, more preferably in a primate, morepreferably in a human.

The term “amino acid composition,” as used herein, refers to the aminoacid sequence and length of a stretch of amino acid residues, whereinthe length is determined by the number of amino acid residues in thatstretch.

The insertion, replacement and/or deletion, preferably insertion, of oneor more amino acid residues can be performed using recombinant DNAtechniques that are well known to the person skilled in the art. Forexample, the person skilled in the art can use synthetic DNA, PCRtechnology and molecular cloning to obtain recombinant DNA constructshaving a DNA sequence encoding a protein of this disclosure. Suitablemethods and means are described in Green and Sambrook, MolecularCloning: A Laboratory Manual, CSHL Press, 2012.

The phrase “corresponding to the region of amino acid residues between,”for example, with regard to the region of amino acid residuescorresponding to the region of amino acid residues between His-311 andAsp-320 of SEQ ID NO: 1,”_([A1]) is used herein to indicate that theresidue number of the conserved His and Asp residues of anothercoagulation FXa corresponding to the His-311 and Asp-320 of SEQ ID NO:1,may differ from the residue number attributed to the His and Asp residuein SEQ ID NO: 1 (see FIGS. 8A-8C). Differences in amino acid residuenumbers can, for example, be the result of a different way of numberingamino acid residues. Also, a difference in amino acid residue number canbe the result of a difference in length of a coagulation FXa polypeptideas compared to the length of the human coagulation FXa polypeptide thatis indicated in FIGS. 8A-8C. Similarly, the amino acid residues Gly-289,Glu-297, Val-305 and Tyr-319, of SEQ ID NO:1, are conserved betweencoagulation FXa polypeptides of different species (see FIGS. 1 and8A-8C). It is, therefore, possible to identify amino acid residues thatcorrespond to the amino acid residues in another coagulation FXapolypeptide. The person skilled in the art will, therefore, understandthat the amino acid residue numbering as applied herein is not limitingfor the disclosure, but is only applied for clarity purposes.

The skilled person will know how to identify a region of amino acidresidues that corresponds to the region of amino acid residues betweenthe conserved amino acid residues of SEQ ID NO:1 that border a region asdescribed herein. When the amino acid residues 289-322 of SEQ ID NO:1are aligned with the corresponding amino acid residues in coagulationFXa polypeptides of different species, it is to be concluded that theamino acid residues at positions 289, 297, 305, 311, 313, 314, 318, 319,320, and 322 of SEQ ID NO:1 are conserved, though not identical, incoagulation FXa polypeptides of different species, especially inmammals, wherein Asp-322 of SEQ ID NO: 1 is a highly conserved catalyticresidue (Asp-102 in chymotrypsinogen numbering; Bode et al., EMBOJournal 1989, 8:3467-3475; Messier et al., Blood Coagulation andFibrinolysis 1996, 7:5-14 and FIGS. 1, 8A, 8B and 8C).

Due to the highly conserved nature of the region of amino acid residuesin and around Gly-289 and Asp-320 of SEQ ID NO: 1, or in and around thecorresponding Gly and Asp residues in a non-human coagulation FXa, theperson skilled in the art is able to identify a region of amino acidresidues corresponding to the region of amino acid residues betweenGly-289 and Asp-320 of SEQ ID NO: 1. The same general principle appliesto other amino acid residues that border a region as described herein.In other words, the conserved nature of specific amino acid residueswill give the skilled person an unambiguous pointer as to which aminoacid residues constitute a region.

A person skilled in the art will understand that this disclosure relatesto the amino acid composition of a region of amino acid residuescorresponding to the region of amino acid residues between Gly-289 andAsp-320, most preferably between His-311 and Asp-320 of SEQ ID NO: 1.Therefore, the person skilled in the art will understand that the aminoacid sequence of the remainder of a protein of the disclosure can vary,under the condition that the protein remains a, or is activated into a,procoagulant FXa polypeptide with decreased sensitivity to DFXIs. Theremainder of a protein of the disclosure may thus vary as it, forexample, varies between coagulation FX, or coagulation FXa, polypeptidesof different species.

The number of amino acid residues in a region corresponding to theregion between Gly-289 and Asp-320, preferably between His-311 andAsp-320 of SEQ ID NO:1 is conserved between coagulation FX proteins ofdifferent species, especially between species belonging to the group ofmammals or to the group of primates. This region is also present inzymogen FX protein and FXa polypeptide. Hence, the number of amino acidresidues is also conserved in zymogen FX protein and FXa polypeptide andin the corresponding region of zymogen FX protein and FXa polypeptide.The conserved number of amino acid residues in a region of amino acidresidues corresponding to the region between Gly-289 and Asp-320 of SEQID NO: 1 is thirty, not including Gly-289 and Asp-320. The conservednumber of amino acid residues in a region of amino acid residuescorresponding to the region between His-311 and Asp-320 of SEQ ID NO: 1is eight, not including His-311 and Asp-320.

It was found that the insertion and/or replacement and/or deletion,preferably the insertion, of at least one amino acid residue in a regionof amino acid residues corresponding to the region of amino acidresidues between Gly-289 and Asp-320, preferably between His-311 andAsp-320 of SEQ ID NO:1 in a protein of the disclosure, yields acatalytically active coagulation FXa with decreased sensitivity toinhibition by DFXIs.

Tyr-319 of human FXa has been demonstrated to be a DFXI-coordinatingresidue (Roehrig et al., J Med. Chem. 2005, 48:5900-5908; Pinto et al.,J. Med. Chem. 2007, 50:5339-5356), while Asp-322 of SEQ ID NO: 1 ispresent in the catalytic serine protease site (Messier et al., BloodCoagulation and Fibrinolysis 1996, 7:5-14). Without being bound bytheory, it is possible that the close proximity of an altered amino acidresidue, such as an insertion of at least one amino acid residue, in theregion between Gly-289 and Asp-320 to the DFXI-coordinating residueTyr-319 of SEQ ID NO:1, or the corresponding tyrosine, and/or the closeproximity to the catalytic domain is responsible for the decreasedsensitivity for a DFXI. It is found that the region of amino acidresidues corresponding to the region of amino acid residues betweenGly-289 and Asp-320 of SEQ ID NO:1 in a protein of the disclosure can bealtered in amino acid residue number and in amino acid sequence, therebygenerating a catalytically active coagulation FXa with decreasedsensitivity for DFXIs.

The alteration is selected from an insertion, a replacement and/or adeletion, and preferably is an insertion, more preferably an insertioncombined with an alteration of at least one amino acid in the regionbetween Gly-289 and Asp-320 of SEQ ID NO: 1.

Particularly preferred is a protein of the disclosure wherein theinsertion is 1-50, preferably 1-20, amino acid residues. The insertionin a region of amino acid residues corresponding to the region of aminoacid residues between Gly-289 and Asp-320, preferably between His-311and Asp-320, of SEQ ID NO:1 in a protein of the disclosure comprises orconsists of between 1-50, preferably between 1-20, amino acid residues.The insertion preferably comprises, or consists of, at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidresidues, resulting in a total of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20, 21, 22, 23, 24, 25, 26, 27, or 28, respectively, amino acidsbetween His-311 and Asp-320. Particularly preferred is the insertion ofat least five amino acid residues, such as an insertion of 9, 12 or 13amino acid residues. The person skilled in the art will understand thatthe amino acid residues can be inserted at any position in a region ofamino acid residues corresponding to the region of amino acid residuesbetween Gly-289 and Asp-320 of SEQ ID NO: 1. An amino acid residuesuitable for insertion is selected from the group of twenty amino acidresidues as listed in Table 1. The person skilled in the art willunderstand that the inserted amino acid residues may undergo apost-translational chemical alteration in vivo or in vitro. As isindicated herein above, the person skilled in the art can use syntheticDNA, PCR technology and molecular cloning to obtain recombinant DNAconstructs having a DNA sequence encoding a protein of this disclosurehaving an insertion of between 1-50 amino acid residues in the region ofamino acid residues corresponding to the region between Gly-289 andAsp-320 of SEQ ID NO:1.

The insertion in a region of amino acid residues corresponding to theregion of amino acid residues between Gly-289 and Asp-320 of SEQ ID NO:1 in a protein of the disclosure is preferably between Thr-315 andLys-316, between Lys-316 and Glu-317, between Glu-317 and Thr-318,and/or between Thr-318 and Tyr-319 of SEQ ID NO:1 or between two aminoacid residues corresponding to these amino acid residues in a non-humancoagulation FXa polypeptide.

Particularly preferred is a protein of the disclosure wherein thereplacement is 1-30, preferably 1-8, more preferably 6 or 7, amino acidresidues. The replacement of amino acid residues in a region of aminoacid residues corresponding to the region of amino acid residues betweenGly-289 and Asp-320 of SEQ ID NO:1 in a protein of the disclosurepreferably comprises, or consists of, between 1-30 amino acid residuesin a region corresponding to the region of amino acid residues betweenGly-289 and Asp-320 of SEQ ID NO:1. The replacement preferablycomprises, or consists of, 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acidresidues. It is preferred that conserved amino acid residues such as,for example, Glu-297, Val-305 and/or His-311 as indicated in SEQ ID NO:1are not replaced. Particularly preferred is the replacement of eithersix or seven amino acid residues.

An amino acid residue present in a region corresponding to the region ofamino acid residues between Gly-289 and Asp-320 of SEQ ID NO:1 of aprotein of the disclosure is preferably replaced by any one of the aminoacid residues listed in Table 1, preferably by an amino acid of the samegroup as is indicated in the columns “side chain polarity” and “sidechain charge” in Table 1. Preferably, one or more of Asn-312, Arg-313,Phe-314, Thr-315, Lys-316, Glu-317, Thr-318 and Tyr-319 of SEQ ID NO:1,or their corresponding amino acid residues in a non-human protein of thedisclosure, are replaced by any one of the amino acid residues asindicated in Table 1. Asn-312 of SEQ ID NO:1 is preferably replaced by aThr or Lys residue. Arg-313 is preferably replaced by an amino acidresidue with a basic polarity and positively charged side-chain (seeTable 1), more preferably by a Lys residue. Amino acid residue Thr-315is preferably replaced by a polar amino acid residue with a neutralside-chain or by a nonpolar amino acid residue with a neutralside-chain, more preferably by a Val residue. Lys-316 of SEQ ID NO:1 ispreferably replaced by a Pro residue. Glu-317 of SEQ ID NO:1 ispreferably replaced by a Val residue. Thr-318 is preferably replaced bya polar amino acid residue with a neutral side-chain or by a nonpolaramino acid residue with a neutral side-chain, more preferably by a Seror Ala residue.

The replacement of amino acid residues in a region of amino acidresidues corresponding to the region of amino acid residues betweenGly-289 and Asp-320 of SEQ ID NO:1 in a protein of the disclosurepreferably comprises, or consists of, at least two amino acid residues.Any combination of at least two amino acid residues is envisaged in thedisclosure, for example, a replacement of Asn-312 of SEQ ID NO: 1 andLys-316 of SEQ ID NO: 1 by a Pro residue and an Ala residue,respectively, or replacement of Asn-312, Arg-313, Thr-315, Lys-316,Glu-317, Thr-318 and Tyr-319 of SEQ ID NO:1 by any one of the amino acidresidues listed in Table 1. Particularly preferred is a protein of thedisclosure having a replacement of (i) Asn-312, (ii) Arg-313, (iii)Thr-315, (iv) Lys-316, (v) Glu-317 and (vi) Thr-318 of SEQ ID NO:1 by a(i) Thr or Pro residue, (ii) Lys residue, (iii) Val residue, (iv) Proresidue, (v) Val residue and (vi) Ser or Ala residue, respectively.

The person skilled in the art will understand that when amino acidresidues are replaced in a region of amino acid residues correspondingto the region between Gly-289 and Asp-320 of SEQ ID NO:1 of a non-humanprotein of the disclosure, only those amino acid residues are replacedthat are not already present in a preferred protein of the disclosure.The person skilled in the art will know that the aforementionedreference to SEQ ID NO:1 is only made in the context of exemplifying thereplacement of amino acid residues in a specified region of amino acidresidues. Therefore, the skilled person will have an indication whichone or more amino acid residues they may replace in a non-humancoagulation FXa for what other amino acid residue or residues.

A protein of the disclosure may further comprise a deletion of at leastone amino acid residue in a region of amino acid residues correspondingto the region of amino acid residues between Gly-289 and Asp-320 of SEQID NO:1. Particularly preferred is a protein of the disclosure having adeletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 or 30 amino acidresidues.

A preferred protein of the disclosure comprises a combination of aninsertion and a replacement, or a combination of an insertion, areplacement, and/or a deletion. Insertions and deletions may occurindependently of each other and it is thus possible that, for example,an insertion of five amino acid residues and a deletion of five aminoacids are present at different amino acid positions in a region of aminoacid residues corresponding to the region of amino acid residues betweenGly-289 and Asp-320 of SEQ ID NO: 1, without affecting the total numberof amino acid residues in a coagulation FX. The skilled person willunderstand that an insertion or deletion changes the amino acid residuenumbering in a protein. With regard to a convenient assessment of wherean alteration is located and what the alteration constitutes, theskilled person can perform a multiple alignment of the amino acidsequence of different coagulation FX proteins as shown in FIGS. 8A-8C.The skilled person can deduce from such an alignment which amino acidresidues are altered. The skilled person can use conserved amino acidresidues, for example, Glu-297, Val-305 and/or His-311 as markers toassess the amino acid residue number where the alteration took place.

Particularly preferred is a protein of the disclosure wherein theinsertion is 1-50, preferably 1-20, amino acid residues and wherein thereplacement is 1-7, preferably 6, amino acid residues. The preferredprotein has an insertion of between 1-50, preferably between 1-20, aminoacid residues, combined with a replacement of between 1-8, preferablyeither 6 or 7, amino acid residues in a region of amino acid residuescorresponding to the region of amino acid residues between Gly-289 andAsp-320 of SEQ ID NO: 1

A more preferred protein of the disclosure has an insertion of at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20amino acid residues and a replacement of at least 1, 2, 3, 4, 5, 6 or 7amino acid residues in a region of amino acid residues corresponding tothe region of amino acid residues between Gly-289 and Asp-320 of SEQ IDNO:1, meaning that the insertion of at least 1-20 amino acid residues iscombined with a replacement of at least 1, 2, 3, 4, 5, 6 or 7 amino acidresidues. The disclosure is directed to all possible combinations of theaforementioned insertion and replacement. Particularly preferred is aprotein having an insertion of 12 or 13 amino acid residues and areplacement of 6 amino acid residues in a region of amino acid residuescorresponding to the region of amino acid residues between Gly-289 andAsp-320 of SEQ ID NO:1.

A protein of the disclosure most preferably comprises a region of aminoacid residues having the amino acid sequence of SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:9, SEQ ID NO: 10 or SEQ ID NO:11 between amino acidresidues corresponding to the amino acid residues His-311 and Asp-320 ofSEQ ID NO:1.

Furthermore, alteration of Arg-366, Glu-369, Phe-396, Asp-413, Ala-414,Cys-415, Gln-416, Ser-419, Val-437, Ser-438, Trp-439, Gly-440, Glu-441,Gly-442, Cys-443, Gly-450, Ile-451 and Tyr-452 of SEQ ID NO: 1 is likelyto result in a protein that is desensitized to DFXIs. Without beingbound by theory, Arg-366, Glu-369, Phe-396, Asp-413, Ala-414, Cys-415,Gln-416, Ser-419, Val-437, Ser-438, Trp-439, Gly-440, Glu-441, Gly-442,Cys-443, Gly-450, Ile-451 and Tyr-452 of SEQ ID NO: 1 are likely to beDFXI-coordinating residues. Literature indirectly supports this view, asit is shown that at least some of these residues are involved in bindingof DFXIs (Roehrig et al., J. Med. Chem. 2005, 48:5900-5908; Pinto etal., J. Med. Chem. 2007, 50:5339-5356). A protein of the disclosurepreferably has replaced or deleted an amino acid residue correspondingto Arg-366, Glu-369, Phe-396, Asp-413, Ala-414, Cys-415, Gln-416,Ser-419, Val-437, Ser-438, Trp-439, Gly-440, Glu-441, Gly-442, Cys-443,Gly-450, Ile-451 or Tyr-452 of SEQ ID NO: 1. Amino acid residuesArg-366, Glu-369, Phe-396, Asp-413, Ala-414, Cys-415, Gln-416, Ser-419,Val-437, Ser-438, Trp-439, Gly-440, Glu-441, Gly-442, Cys-443, Gly-450,Ile-451 and/or Tyr-452 of SEQ ID NO:1, or the corresponding amino acidresidues in a related protein, are preferably replaced by any one of theamino acid residues as listed in Table 1. Also, a protein of thedisclosure preferably has an insertion of at least one amino acidresidue, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 amino acid residues in a region of amino acid residuescorresponding to the region between the 15 amino acid residues locatedat the N-terminal and 15 amino acid residues located at the C-terminalfrom Arg-366, Glu-369, Phe-396, Asp-413, Ala-414, Cys-415, Gln-416,Ser-419, Val-437, Ser-438, Trp-439, Gly-440, Glu-441, Gly-442, Cys-443,Gly-450, Ile-451 and/or Tyr-452. The alteration of Phe-396, Arg-366,Glu-369, Asp-413, Ala-414, Cys-415, Gln-416, Ser-419, Val-437, Ser-438,Trp-439, Gly-440, Glu-441, Gly-442, Cys-443, Gly-450, Ile-451 and/orTyr-452 of SEQ ID NO:1. The insertion in a region as indicated in thisparagraph is preferably combined with an alteration in the regionbetween Gly-289 and Asp-320 of SEQ ID NO:1 as defined hereinabove.

This disclosure also encompasses proteins that are substantiallyhomologous and biologically equivalent to a protein of the disclosure. Aprotein of the disclosure preferably has an amino acid sequence that ismore than 60%, preferably more than 70%, more preferably more than 80%,and most preferably more than 90% homologous to SEQ ID NO:1 or to theactivated form thereof, wherein the protein is catalytically active(procoagulant), or catalytically active after processing/activation, andhas a decreased sensitivity to DFXIs, preferably a DFXI selected fromthe group consisting of rivaroxaban, apixaban, edoxaban and betrixaban.The person skilled in the art knows how the preproprotein or proproteinof coagulation FX is processed to its catalytically active form. TheUniProt database provides an overview, under Accession Number P00742, ofthe processing of human coagulation FX to activated human coagulationFXa. The skilled person will thus be able to determine which amino acidresidues are present or absent in coagulation FXa.

The term “decreased sensitivity to DFXIs,” as used in the context ofthis disclosure, refers to the concentration of a DFXI that is requiredto produce 50% of the maximum inhibition (Ki), that is higher for apolypeptide of this disclosure than for a native coagulation FXa,wherein the native coagulation FXa is preferably derived from bloodplasma or is recombinantly produced. The Ki of a DFXI is preferablydetermined by pre-incubating a protein of the disclosure with 0.001 to100 μM of a DFXI and subsequently performing an experiment wherein thecatalytic activity toward Spectrozyme Xa (Sekisui Diagnostics; Stamford,Conn., USA) by peptidyl substrate conversion is assayed. The Ki of aprotein of the disclosure is preferably increased more than two times,more preferably, increased between 50 and 100 times, and mostpreferably, increased more than 100 times as compared to the Ki of thenative coagulation FXa without an alteration of at least one amino acidresidue in a region of amino acid residues corresponding to the regionof amino acid residues between Gly-298 and Asp-320 of SEQ ID NO: 1.

It was unexpectedly found that a protein of the disclosure has anincreased binding affinity for coagulation FVa, the binding partner ofcoagulation FXa in the prothrombinase complex, as compared to thebinding affinity of native coagulation FXa for coagulation FVa. Thebinding affinity of a human or humanized protein of the disclosure forFVa is at least two times higher than the binding affinity of nativehuman FXa for FVa.

Assays for determining the binding affinity are known in the art, forexample, by using a binding partner (such as FVa or FXa) with aradiolabel. The amount of radiation emitted upon binding can be used tocalculate the binding affinity. Also, non-radioactive methods such assurface plasmon resonance and dual polarization interferometry can beused to quantify the binding affinity from concentration-based assaysbut also from the kinetics of association and dissociation and, in thelatter, the conformational change induced upon binding. Recently,Microscale Thermophoresis (MST), an immobilization-free method wasdeveloped, that allows the determination of the binding affinity betweentwo proteins (Wienken et al., Nature Communications 2010, 1:100).Preferably, the binding affinity of the coagulation FVa-FXa complex isdetermined via either the kinetics of prothrombin or prothrombinderivatives (prethrombin-1, prethrombin-2) conversion (Bos et al., Blood2009, 114:686-692), fluorescence intensity/anisotropy measurements (Boset al., J. Biol. Chem. 2012, 287: 26342-51), or isothermal titrationcalorimetry (ITC).

The disclosure further provides a nucleic acid molecule comprising a DNAsequence that encodes a protein of the disclosure. The person skilled inthe art will understand how to generate a DNA sequence that encodes anamino acid sequence of a protein of this disclosure and how tomanufacture and isolate a nucleic acid molecule with the DNA sequenceusing generally known recombinant DNA techniques. The sequence of thenucleic acid molecule is preferably codon-optimized for expression in ahost cell of the disclosure. In this way, codons are used that arefavored for high-level expression in a specific host cell.

This disclosure also provides an expression vector comprising a nucleicacid molecule of the disclosure.

Nucleic acid molecules are preferably inserted in an expression vectorusing recombinant DNA techniques known by the person skilled in the art.Expression vectors in the context of the disclosure direct theexpression of a protein of the disclosure in a host cell. Theseexpression vectors are preferably replicable in a host cell, either asepisomes or as part of the chromosomal DNA. Further, the expressionvector preferably comprises (i) a strong promoter/enhancer, such as theCMV or SV40 promoter, (ii) an optimal translation initiation sequence,such as a ribosomal binding site and start codon, preferably a KOZAKconsensus sequence and (iii) a transcription termination sequence,including a poly(A) signal when the protein is expressed in eukaryoticcells. Suitable expression vectors include plasmids and viral vectorssuch as adenoviruses, adeno-associated viruses and retroviruses. Theperson skilled in the art will understand that the expression vector tobe used is dependent on the host cell that is used for expression of arecombinant protein. An expression vector of the disclosure ispreferably suited for expression of a nucleic acid molecule of thedisclosure in a prokaryotic cell including a bacterial cell, or, morepreferred, in a eukaryotic host cell, such as a yeast cell and amammalian cell. Particularly preferred is mammalian expression vectorpCMV4.

As an alternative, a nucleic acid molecule of the disclosure may beinserted in the genome of a host cell. The insertion preferably is at alocus or within a region that ensures expression of a nucleic acidmolecule of the disclosure in the host cell.

The disclosure further provides a host cell comprising a nucleic acidmolecule of the disclosure. The disclosure preferably provides a hostcell expressing a nucleic acid molecule of the disclosure, therebyproducing a protein of the disclosure. The protein is either producedwithin the host cell or, preferably, secreted from the host cell.

Suitable host cells for use in this disclosure include prokaryotic andeukaryotic cells, such as bacterial cells, yeast cells, insect cells,animal cells, mammalian cells, murine cells, rat cells, sheep cells,simian cells and human cells. Examples of suitable eukaryotic host cellsinclude, but are not limited to, HEK 293 cells, the hamster cell lineCHO and BHK-21; the murine host cells NIH3T3, NSO and C127; the simianhost cells COS and Vero; and the human host cells HeLa, PER.C6®, U-937and Hep G2. Suitable cells are available from public sources such asATCC and Life Technologies. A number of transfection techniques areknown in the art, see, e.g., Graham et al., Virology 1973, 52:456; Greenet al., Molecular Cloning: A Laboratory Manual 2012, CSHL Press; Daviset al., Basic Methods in Molecular Biology 1986, Elsevier; and Chu etal., Gene 1981, 13:197. The person skilled in the art preferably employstechniques as described in these references to introduce one or moreexogenous nucleic acid molecules into suitable host cells.

A particularly preferred host cell for the production of a protein ofthe disclosure is a HEK 293 cell.

The disclosure further provides a pharmaceutical composition comprisinga protein of the disclosure, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier or excipient. Apharmaceutical composition of the disclosure preferably comprises one ormore of diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials known in the art. The characteristics of the carrierwill depend on the route of administration, as is known to the skilledperson. To reduce the potential thrombotic risk of administering theserine protease FXa, a pharmaceutical composition of the disclosurepreferably comprises a protein of the disclosure that is activated afteradministering to the subject.

The term “subject” refers to the group of mammals, preferably humans.

The term “pharmaceutical composition” refers, in the context of theinvention, to a combination of a protein of the disclosure with acarrier, inert or active, making the composition suitable fortherapeutic use in vivo or ex vivo.

The term “pharmaceutically acceptable,” as used herein, refers to anontoxic material that is compatible with the physical and chemicalcharacteristics of a protein of the disclosure and does not interferewith the effectiveness of the biological activity of the protein.

A pharmaceutical composition of the disclosure may be adapted forenteral administration of the composition, wherein the composition isabsorbed through the digestive tract, e.g., oral ingestion or rectaladministration. The composition is preferably encapsulated, for example,by liposomes, to prevent proteolytic degradation.

A pharmaceutical composition of the disclosure preferably is appliedlocally, for example, at or in a wound or to a blood vessel, preferablyan artery, that supplies the wounded region with blood. The localadministration is a topical administration, for example, in the form ofa cream, foam, gel, lotion or ointment, or a parenteral administration,for example, by injection or infusion, to generate a local or systemictherapeutic effect. Topical administration of a protein of thedisclosure for a local effect reduces the risk of a potential systemicthrombotic incident.

A pharmaceutical composition of the disclosure, preferably comprisingcoagulation FX or a mature coagulation FX that comprises an alteredcoagulation factor Xa polypeptide, is preferably systemicallyadministered, preferably by parenteral administration. Systemicadministration of an inactive preproprotein or inactive proprotein willresult in the formation of an active prothrombinase complex thatconsists of coagulation FXa in association with FVa on negativelycharged phospholipid membranes where it converts inactive prothrombininto the active serine protease thrombin.

A pharmaceutical composition of the disclosure is preferably adapted forparenteral administration, wherein the composition is intravenously,intra-arterial, subcutaneously, and/or intramuscularly introduced.Parenteral administration involves the injection or infusion of apharmaceutical composition of the disclosure into a body tissue or bodyfluid, whereby preferably a syringe, needle, or catheter is used. As analternative, needle-less high-pressure administration may be used asmeans for parenteral administration.

For injectable compositions (e.g., intravenous compositions), thecarrier may be aqueous or oily solutions, dispersions, emulsions and/orsuspensions. Preferably, the carrier is an aqueous solution, preferablydistilled sterile water, saline, buffered saline, or anotherpharmaceutically acceptable excipient for injection.

A pharmaceutical composition of the disclosure is preferably used in avariety of therapeutical applications. For example, the pharmaceuticalcomposition can be used as bypassing agent in the treatment oramelioration of disorders wherein normal blood coagulation is impaired,such as in hemophilia A and B, including in hemophilia A and B inhibitorpatient groups, or in factor X deficiency.

The disclosure further provides a protein according to the disclosure orpharmaceutical composition according to the disclosure for use in amethod of completely or partially reversing an anti-coagulant effect ofa coagulation inhibitor in a subject.

The term “anti-coagulant effect” refers to the therapeutic effect, suchas the prevention of blood clotting, that is the result of the action ofcoagulation inhibitors.

The disclosure further provides the use of a protein of the disclosurefor the manufacture of a medicament for completely or partiallyreversing an anti-coagulant effect of a coagulation inhibitor in asubject.

The coagulation inhibitor preferably is a direct FXa inhibitor (DFXI),more preferably a direct FXa inhibitor selected from the group formed byrivaroxaban(5-chloro-N-[[(5S)-2-oxo-3-[4-(3-oxo-4-morpholinyl)phenyl]-5-oxazolidinyl]methyl]-2-thiophenecarboxamide),apixaban(1-(4methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide),edoxaban(N′-(5-chloropyridin-2-yl)-N-[(1S,2R,4S)-4-(dimethylcarbamoyl)-2-[(5-methyl-6,7-dihydro-4H-[1,3]thiazolo[5,4-c]pyridine-2-carbonyl)amino]cyclohexyl]oxamide;4-methylbenzenesulfonic acid) and/or betrixaban(N-(5-chloropyridin-2-yl)-2-[[4-(N,N-dimethylcarbamimidoyl)benzoyl]amino]-5-methoxybenzamide).

The disclosure further provides a method of completely or partiallyreverting an anti-coagulant effect of a coagulation inhibitor in asubject, the method comprising administering to the subject atherapeutically effective amount of a protein of the disclosure or apharmaceutical composition of the disclosure. Preferably, a method ofthe disclosure is applied for preventing or ameliorating bleedingcomplications that are associated with anticoagulant therapy.

The term “therapeutically effective amount” as used herein means thatthe amount of the active ingredient contained in the pharmaceuticalcomposition to be administered is of sufficient quantity to achieve theintended purpose, such as, in this case, to completely or partiallyreverse an anti-coagulant effect of a coagulation inhibitor. The amountof active ingredient, i.e., a protein of the disclosure, in apharmaceutical composition according to the disclosure preferably is inthe range of about 50 mg to about 600 mg. A pharmaceutical compositionaccording to the disclosure is preferably administered only once, twiceor three times, preferably only once, to a subject in need of completeor partial reversal of an anti-coagulant effect of a coagulationinhibitor.

For the purpose of clarity and a concise description, features aredescribed herein as part of the same or separate embodiments; however,it will be appreciated that the scope of the disclosure may includeembodiments having combinations of all or some of the featuresdescribed.

(human coagulation factor X protein) SEQ ID NO: 1   1mgrplhlvll saslagllll geslfirreq annilarvtr ansfleemkk ghlerecmee  61tcsyeearev fedsdktnef wnkykdgdqc etspcqnqgk ckdglgeytc tclegfegkn 121celftrklcs ldngdcdqfc heeqnsvvcs cargytladn gkaciptgpy pcgkqtlerr 181krsvaqatss sgeapdsitw kpydaadldp tenpfdlldf nqtqpergdn nltrivggqe 241ckdgecpwqa llineenegf cggtilsefy iltaahclyq akrfkvrvgd rnteqeegge 301avhevevvik hnrftketyd fdiavlrlkt pitfrmnvap aclperdwae stlmtqktgi 361vsgfgrthek grqstrlkml evpyvdrnsc klsssfiitq nmfcagydtk qedacqgdsg 421gphvtrfkdt yfvtgivswg egcarkgkyg iytkvtaflk widrsmktrg lpkakshape 481vitssplk (Light chain of human coagulation factor Xa) SEQ ID NO: 2   1ansfleemkk ghlerecmee tcsyeearev fedsdktnef wnkykdgdqc etspcqnqgk  61ckdglgeytc tclegfegkn celftrklcs ldngdcdqfc heeqnsvvcs cargytladn 121gkaciptgpy pcgkqtler 139 (Heavy chain of human coagulation factor Xa)SEQ ID No 3   1ivggqeckdg ecpwqallin eenegfcggt ilsefyilta ahclyqakrf kvrvgdrnte  61qeeggeavhe vevvikhnrf tketydfdia vlrlktpitf rmnvapaclp erdwaestlm 121tqktgivsgf grthekgrqs trlkmlevpy vdrnscklss sfiitqnmfc agydtkqeda 181cqgdsggphv trfkdtyfvt givswgegca rkgkygiytk vtaflkwidr smktrglpka 241kshapevits splk SEQ ID NO: 4   1 tkfvppnyyyvhqnfdrvay SEQ ID NO: 5   1kkfvppkksclefyekfdlvsy (human coagulation FX gene; 1-1473 bp)SEQ ID NO: 6atggcgcacgtccgaggcttgcagctgcctggctgcctggccctggctgccctgtgtagccttgtgcacagccagcatgtgttcctggctcctcagcaagcacggtcgctgctccagcgggtccggcgagccaattcctttcttgaagagatgaagaaaggacacctcgaaagagagtgcatggaagagacctgctcatacgaagaggcccgcgaggtctttgaggacagcgacaagacgaatgaattctggaataaatacaaagatggcgaccagtgtgagaccagtccttgccagaaccagggcaaatgtaaagacggcctcggggaatacacctgcacctgtttagaaggattcgaaggcaaaaactgtgaattattcacacggaagctctgcagcctggacaacggggactgtgaccagttctgccacgaggaacagaactctgtggtgtgctcctgcgcccgcgggtacaccctggctgacaacggcaaggcctgcattcccacagggccctacccctgtgggaaacagaccctggaacgcaggaagaggtcagtggcccaggccaccagcagcagcggggaggcccctgacagcatcacatggaagccatatgatgcagccgacctggaccccaccgagaaccccttcgacctgcttgacttcaaccagacgcagcctgagaggggcgacaacaacctcacgcgtatcgtgggaggccaggaatgcaaggacggggagtgtccctggcaggccctgctcatcaatgaggaaaacgagggtttctgtggtggaactattctgagcgagttctacatcctaacggcagcccactgtctctaccaagccaagagattcaaggtgagggtaggtgaccggaacacggagcaggaggagggcggtgaggcggtgcacgaggtggaggtggtcatcaagcacaaccggttcacaaaggagacctatgacttcgacatcgccgtgctccggctcaagacccccatcaccttccgcatgaacgtggcgcctgcctgcctccccgagcgtgactgggccgagtccacgctgatgacgcagaagacggggattgtgagcggcttcgggcgcacccacgagaagggccggcagtccaccaggctcaagatgctggaggtgccctacgtggaccgcaacagctgcaagctgtccagcagcttcatcatcacccagaacatgttctgtgccggctacgacaccaagcaggaggatgcctgccagggggacagcgggggcccgcacgtcacccgcttcaaggacacctacttcgtgacaggcatcgtcagctggggagagggctgtgcccgtaaggggaagtacgggatctacaccaaggtcaccgccttcctcaagtggatcgacaggtccatgaaaaccaggggcttgcccaaggccaagagccatgccccggaggtcataacgtcctctccattgaaa(DNA sequence of modified human FX-type A; 1-1509 bp) SEQ ID NO: 7Atggcgcacgtccgaggcttgcagctgcctggctgcctggccctggctgccctgtgtagccttgtgcacagccagcatgtgttcctggctcctcagcaagcacggtcgctgctccagcgggtccggcgagccaattcctttcttgaagagatgaagaaaggacacctcgaaagagagtgcatggaagagacctgctcatacgaagaggcccgcgaggtctttgaggacagcgacaagacgaatgaattctggaataaatacaaagatggcgaccagtgtgagaccagtccttgccagaaccagggcaaatgtaaagacggcctcggggaatacacctgcacctgtttagaaggattcgaaggcaaaaactgtgaa.ttattcacacggaagctctgcagcctggacaacggggactgtgaccagttctgccacgaggaacagaactctgtggtgtgctcctgcgcccgcgggtacaccctggctgacaacggcaaggcctgcattcccacagggccctacccctgtgggaaacagaccctggaacgcaggaagaggtcagtggcccaggccaccagcagcagcggggaggcccctgacagcatcacatggaagccatatgatgcagccgacctggaccccaccgagaaccccttcgacctgcttgacttcaaccagacgcagcctgagaggggcgacaacaacctcacgcgtatcgtgggaggccaggaatgcaaggacggggagtgtccctggcaggccctgctcatcaatgaggaaaacgagggtttctgtggtggaactattctgagcgagttctacatcctaacggcagcccactgtctctaccaagccaagagattcaaggtgagggtaggtgaccggaacacggagcaggaggagggcggtgaggcggtgcacgaggtggaggtggtcatcaagcacaccaagttcgtgccccctaactactattacgt ccaccagaattttgaccgggtggcctatgacttcgacatcgccgtgctccggctcaagacccccatcaccttccgcatgaacgtggcgcctgcctgcctccccgagcgtgactgggccgagtccacgctgatgacgcagaagacggggattgtgagcggcttcgggcgcacccacgagaagggccggcagtccaccaggctcaagatgctggaggtgccctacgtggaccgcaacagctgcaagctgtccagcagcttcatcatcacccagaacatgttctgtgccggctacgacaccaagcaggaggatgcctgccagggggacagcgggggcccgcacgtcacccgcttcaaggacacctacttcgtgacaggcatcgtcagctggggagagggctgtgcccgtaaggggaagtacgggatctacaccaaggtcaccgccttcctcaagtggatcgacaggtccatgaaaaccaggggcttgcccaaggccaagagccatgccccggaggtcataacgtcctctccattgaaa (DNA sequence of modified human FX-type B; 1-1512 bp)SEQ ID NO: 8atggcgcacgtccgaggcttgcagctgcctggctgcctggccctggctgccctgtgtagccttgtgcacagccagcatgtgttcctggctcctcagcaagcacggtcgctgctccagcgggtccggcgagccaattcctttcttgaagagatgaagaaaggacacctcgaaagagagtgcatggaagagacctgctcatacgaagaggcccgcgaggtctttgaggacagcgacaagacgaatgaattctggaataaatacaaagatggcgaccagtgtgagaccagtccttgccagaaccagggcaaatgtaaagacggcctcggggaatacacctgcacctgtttagaaggattcgaaggcaaaaactgtgaattattcacacggaagctctgcagcctggacaacggggactgtgaccagttctgccacgaggaacagaactctgtggtgtgctcctgcgcccgcgggtacaccctggctgacaacggcaaggcctgcattcccacagggccctacccctgtgggaaacagaccctggaacgcaggaagaggtcagtggcccaggccaccagcagcagcggggaggcccctgacagcatcacatggaagccatatgatgcagccgacctggaccccaccgagaaccccttcgacctgcttgacttcaaccagacgcagcctgagaggggcgacaacaacctcacgcgtatcgtgggaggccaggaatgcaaggacggggagtgtccctggcaggccctgctcatcaatgaggaaaacgagggtttctgtggtggaactattctgagcgagttctacatcctaacggcagcccactgtctctaccaagccaagagattcaaggtgagggtaggtgaccggaacacggagcaggaggagggcggtgaggcggtgcacgaggtggaggtggtcatcaagcacaagaaattcgtgccccctaagaaaagccaggag ttctacgaaaagtttgacctggtctcctatgacttcgacatcgccgtgctccggctcaagacccccatcaccttccgcatgaacgtggcgcctgcctgcctccccgagcgtgactgggccgagtccacgctgatgacgcagaagacggggattgtgagcggcttcgggcgcacccacgagaagggccggcagtccaccaggctcaagatgctggaggtgccctacgtggaccgcaacagctgcaagctgtccagcagcttcatcatcacccagaacatgttctgtgccggctacgacaccaagcaggaggatgcctgccagggggacagcgggggcccgcacgtcacccgcttcaaggacacctacttcgtgacaggcatcgtcagctggggagagggctgtgcccgtaaggggaagtacgggatctacaccaaggtcaccgccttcctcaagtggatcgacaggtccatgaaaaccaggggcttgcccaaggccaagagccatgccccggaggtcataacgtcctctccattgaaa SEQ ID NO: 9   1 kkfvppkksqefyekfdlaay SEQ ID NO: 10   1kkfvppnyyyvhqnfdlaay SEQ ID NO: 11   1 kkfvppqkaykfdlaay

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Blood coagulation FXa structure. Panel A: Schematicγ-carboxyglutamic (“GLA”) EGF-1 and -2 (“EGF”), and serine proteasedomain (“SP”) structure of coagulation FXa. Panel B: Crystal structureof the human FXa serine protease (pdb 2W26). Indicated are the catalytictriad His-276, Asp-322, Ser-419, rivaroxaban/apixaban contact residuesTry-319 and Phe-396, position of residues 316-317 (in spheres), andresidues Gly-289, Glu-297, Val-305, and His-311. Panel C: Alignment ofregion 311-322 in various plasma FX species with conserved residues(highlighted), contact residue Tyr-319, and catalytic residue Asp-322indicated (SEQ ID NOS. 1 and 29-36). *Indicates venom coagulation FXwith insertion in the region corresponding to the region between Gly-289and Asp-320 of SEQ ID NO:1.

FIGS. 2A-2C: Inhibition of chromogenic FXa activity by direct FXainhibitors. FIG. 2A: Peptidyl substrate conversion (SpecFXa, 250 riM) byrecombinant human coagulation FXa (hFXa, 2 nM, circles) or venom P.textilis coagulation FXa (vptFXa, 10 nM, triangles) in the presence ofincreasing concentrations (1 nM-10 μM) rivaroxaban (“riva,” closedsymbols) or apixaban (“api,” open symbols). Substrate conversion isplotted as the % of incubations in the absence of inhibitor. FIGS. 2Band 2C: Thrombin generation in coagulation FX-deficient plasma wasinitiated with 0.5 nM hFXa (FIG. 2B) or vptFXa (FIG. 2C) in the absence(grey line/grey column) or presence of 0.4 μM rivaroxaban (“riva,” blackline/black column) or 2 μM apixaban (“api,” dotted line/white column).Thrombin formation was assessed using a fluorogenic substrate and peakthrombin concentrations of the various incubations are shown in theinsets.

FIGS. 3A and 3B: FIG. 3A: Fluorescent Western blot of recombinant FX(200 ng) obtained from HEK293 cell lines that stably express eitherrecombinant human FX (r-hFX, lane 1, 5), modified human FX-A (mod A,lane 2, 6) or modified human FX-B (mod B, lane 3, 7), before (lanes 1,2, 3) or after (lanes 5, 6, 7) incubation with RVV-X activator. Theheavy chain of endogenous plasma-derived human FXa migrates at ˜29 kDa(lane 9). Relative weight (kDa) of the protein markers (lanes 4, 8) areindicated. FIG. 3B: Recombinant FX in conditioned media from HEK293 celllines stably expressing either recombinant human FX (black column),modified human FX-A (white column) or modified human FX-B (grey column)was quantified using an FX-specific ELISA. Each individual barrepresents a single stable cell line with the highest attainableexpression per FX variant.

FIG. 4: Macromolecular substrate activation. Prothrombin conversion (1.4μM) in the presence of 50 μM PCPS, 20 nM FV (FV810, recombinant B-domaintruncated FV) and 0.1 nM of modified human coagulation FXa type A(m-hFXa A), type B (m-hFXa B), recombinant (r-hFXa), or plasma-derived(pd-hFXa) FXa. The substrate conversion is plotted in nM/min/nM Enzymeand data are the mean value of two independent experiments+S.D.

FIGS. 5A and 5B: Inhibition of FXa chimer type-A by DFXIs. Peptidylsubstrate conversion (SpecFXa, 250 μM) of RVV-X activated modified humancoagulation FXa type A (m-hFXa A, 1 nM) in comparison to recombinanthuman coagulation FXa (r-hFXa, 3 nM), plasma-derived human coagulationFXa (pd-FXa, 2 nM), and venom P. textilis (vptFXa, 1 nM) FXa. Conversionrates were determined in the presence of 0.001-100 μM of Rivaroxaban(FIG. 5A) or Apixaban (FIG. 5B). The data represent the mean value oftwo independent experiments, except for r-hFXa (n=1).

FIGS. 6A and 6B: Inhibition of modified human FX-A and modified humanFX-B by DFXIs. Peptidyl substrate conversion (SpecFXa, 250 μM) by RVV-Xactivated modified human FX-A (m-hFXa A, 1 nM) and modified human FX-B(m-hFXa B, 7 nM, in comparison to RVV-X-activated recombinant humancoagulation FXa (r-hFXa, 6 nM). Conversion rates were determined in thepresence of 0.001-100 μM of rivaroxaban (FIG. 6A) and apixaban (FIG.6B). The data are the means of two independent experiments.

FIGS. 7A and 7B: Inhibition of modified human FX-A or modified humanFX-B by DFXIs in the presence of cofactor Va and phospholipids. Peptidylsubstrate conversion (SpecFXa, 250 μM) by RVV-X activated modified humanFX-A (m-hFXa A, 2 nM) and activated modified human FX-B (m-hFXa B, 4 nM)in comparison to by RVV-X activated recombinant human coagulation FXa(r-hFXa, 3 nM), in the presence of 50 μM PCPS and 30 nM FV (FV810,recombinant B-domain truncated). Conversion rates were determined in thepresence of 0.001-100 μM of Rivaroxaban (FIG. 7A) or Apixaban (FIG. 7B).The data are the means of two independent experiments.

FIGS. 8A-8C: Multiple alignment of coagulation FX proteins of differentspecies. The amino acid sequence of human coagulation FX (GenbankAccession No.: AAH46125.1) (HUM) (SEQ ID NO:1) is compared to the aminoacid sequences of M. musculus coagulation FX (Genbank Accession No.:AAC36345.1) (MUS) (SEQ ID NO:29), X. tropicalis coagulation FX (GenbankAccesion No.: NP_001015728) (Xtr) (SEQ ID NO:30), D. rerio coagulationFX (Genbank Accession No.: AAM88343.1) (Dre) (SEQ ID NO:31), T. rubripescoagulation FX (Genbank Accession No.: NP_001027783.1) (Tru) (SEQ IDNO:32), P. textilis coagulation FX isoform 1 (UniprotKB accession No.:Q1L659) (Pte1) (SEQ ID NO:33), P. textilis coagulation FX isoform 2(UniprotKB accession No.: Q1L658) (Pte2) (SEQ ID NO:34), P. textiliscoagulation FX (pseutarin C catalytic subunit precursor; GenbankAccession No.: AAP86642.1) (Pte3) (SEQ ID NO:35) and N. scutatuscoagulation FX (UniProtKB accession No.: P82807.2) (Nsc) (SEQ ID NO:36).In these figures, Gly-289, Asp-320, Tyr-319, Glu-297, Val-305 andHis-311 of SEQ ID NO:1 are indicated in bold and are underlined. Thesefigures show that there is variation in the region of amino acidresidues corresponding to the region between Gly-289 and Asp-320 of SEQID NO:1 between coagulation FX proteins of different species. Amino acidresidues that are conserved in all species are indicated in theconsensus sequence.

FIG. 9: Amino acid composition of endogenous hFX and chimeric FXvariants. Serine protease domain residues Histidine91 and Tyrosine99(chymotrypsin numbering; corresponding to His 311 and Tyrosine 319,respectively, of FX as depicted in SEQ ID NO: 1) of endogenous human(hFX) in alignment with chimeric FX type A (c-FX A, middle; sequencebetween His 311 and Asp 320 corresponds to SEQ ID NO:9), type B (c-FX B;sequence between His 311 and Asp 320 corresponds to SEQ ID NO:10), andtype C (c-FX C; sequence between His 311 and Asp 320 corresponds to SEQID NO: 11).

FIG. 10: Characterization of FXa: Panel A: Coomassie staining of 5 μgFXa variants on 4-12% Bis-Tris gels. From left to right: plasma-derivedFactor Xa (pd-FXa), r-hFXa, chimeric factor Xa type A, B and C (-A, -B,-C). Panel B: Prothrombin conversion (1.4 μM) in the presence of 50 μMPCPS (75% phosphatidylcholine, 25% phosphatidylserine) and 20 nM FV(FV810, recombinant B-domain truncated FV) and 0.1 nM of pd-FXa, r-hFXa,c-FXa-A, c-FXa-B and c-FXa-C. Data points are the mean value of twoindependent experiments.

FIG. 11: Inhibition of FXa variants by DOACs. Normalized prothrombinconversion by 1 nM of pd-FXa (triangles), r-hFXa (circles), chimericFXa-A (squares), -B (diamonds) and -C (crosses) was assessed in thepresence of 0.001-100 μM of Apixaban (left, closed symbols) or Edoxaban(right, open symbols). Inhibitory constants (determined with GraphpadPrism 6 software suite) of Apixaban for pd-FXa: 2 nM, r-hFXa: 4 nM,c-FXa-A: 130 nM, -B: 760 nM-C: 1270 nM and of Edoxaban for r-hFXa: 0.5nM, c-FXa-A: 3 nM, -B: 140 nM-C: 270 nM.

FIGS. 12A and 12B: FXa-initiated thrombin generation (TG) profiles forFXa variants. Plasma TG in the absence (FIG. 12A) and presence (FIG.12B) of DOAC Apixaban (2 μM). Initiation of TG by pd-FXa, r-hFXa,c-FXa-A, c-FXa-B and c-FXa-C in FX-depleted plasma. Curves are theaverage of at least three independent experiments.

FIGS. 13A and 13B: Tissue factor (TF)-initiated TG profile for r-hFX andc-FX-C. FIG. 13A: Plasma TG at low TF (2 μM) in the absence and presenceof 2 μM DOAC Apixaban (Apixa) by 1 unit r-hFX, r-hFX plus Apixaban,c-FXa-C or c-FXa-C plus Apixaban. One unit of r-hFX (7 μg/ml) or c-FXa-C(16 μg/ml) was defined by a prothrombin time-based clotting assay usingnormal human plasma as reference. Curves represent the average of atleast three independent experiments. FIG. 13B: Plasma TG at high TF (20μM).

FIGS. 14A and 14B: TF-initiated TG profile for r-hFX and c-FX-C. (Uppergraph): Plasma TG at low TF (2 μM) in the absence (dotted line) andpresence of 200 nM (light grey), 600 nM (dark grey) and 2000 nM (black)DOAC Edoxaban by 1 unit r-hFX (7 μg/ml). (Lower graph): Plasma TG at lowTF (2 μM) with similar concentrations of Edoxaban by 1 unit of c-FXa-C(16 μg/ml). Curves represent the average of two independent experiments.

TABLE 1 Side-chain Side-chain charge Hydropathy Absorbance ε at λmax(×10⁻³ M⁻¹ Amino Acid 3-Letter⁽¹¹⁴⁾ 1-Letter⁽¹¹⁴⁾ polarity⁽¹¹⁴⁾ (pH7.4)⁽¹¹⁴⁾ Index⁽¹¹⁵⁾ λmax(nm)⁽¹¹⁶⁾ cm⁻¹)⁽¹¹⁶⁾ Alanine Ala A nonpolarneutral 1.8 Arginine Arg R Basic polar positive −4.5 Asparagine Asn Npolar neutral −3.5 Aspartic acid Asp D acidic polar negative −3.5Cysteine Cys C nonpolar neutral 2.5 250 0.3 Glutamic acid Glu E acidicpolar negative −3.5 Glutamine Gln Q polar neutral −3.5 Glycine Gly Gnonpolar neutral −0.4 Histidine His H Basic polar positive (10%) −3.2211 5.9 neutral (90%) Isoleucine Ile I nonpolar neutral 4.5 Leucine LeuL nonpolar neutral 3.8 Lysine Lys K Basic polar positive −3.9 MethionineMet M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar neutral 2.8 257,200, 188 0.2, 9.3, 00.0 Proline Pro P nonpolar neutral −1.6 Serine Ser Spolar neutral −0.8 Threonine Thr T polar neutral −0.7 Tryptophan Trp Wnonpolar neutral −0.9 280, 219 5.6, 47.0 Tyrosine Tyr Y polar neutral−1.3 274, 222, 193 1.4, 8.0, 48.0 Valine Val V nonpolar neutral 4.2

DETAILED DESCRIPTION Examples Example 1

Materials and Methods

Rivaroxaban and Apixaban were obtained from Alsachim (Illkirch, France)and dissolved in DMSO (˜30 mg/ml). The peptidyl substratemethoxycarbonylcyclohexylglycylglycylarginine-p-nitroanilide (Spec-Xa)was obtained from Sekisui Diagnostics (Stamford, Conn., USA). All tissueculture reagents were from Life Technologies (Carlsbad, Calif.), exceptinsulin-transferrin-sodium selenite (ITS), which was from Roche (Basel,Switzerland). Small unilamellar phospholipid vesicles (PCPS) composed of75% (w/w) hen egg L-phosphatidylcholine and 25% (w/w) porcine brainL-phosphatidylserine (Avanti Polar Lipids, Alabaster, Ala.) wereprepared and characterized as described previously (Higgins et al., J.Biol. Chem. 1983, 258:6503-6508). FX-depleted human plasma was obtainedfrom Diagnostica Stago (Paris, France). All functional assays wereperformed in HEPES buffered Saline (20 mM Hepes, 0.15 M NaCl, pH 7.5)supplemented with 5 mM CaCl2 and 0.1% polyethylene glycol 8000 (assaybuffer). Mammalian expression vector pCMV4 (Andersson et al., J. Biol.Chem. 1989, 264:8222-8229, carrying recombinant human FX (r-hFX) was agenerous gift from Rodney M. Camire (Camire et al., Biochemistry 2000,39:14322-14329). The pcDNA3 vector was obtained from Invitrogen and thePACE cDNA was a generous gift from Genetics Institute, Boston, Mass. Avector carrying Furin proprotein convertase has been described (U.S.Pat. No. 5,460,950).

Human recombinant Factor V (FV) was prepared, purified, andcharacterized as described previously (Bos et al., Blood 2009,114:686-692). Recombinant P. textilis venom FXa (vpt-FXa) was prepared,purified, and characterized as described previously (Verhoef et al.,Toxin Reviews (2013) (doi:10.3109/15569543.2013.844712). Plasma-derivedhuman Factor Xa (pd-hFXa), DAPA, human prothrombin and Anti-Human FactorX monoclonal mouse IgG (AHX-5050) were from Haematologic Technologies(Essex Junction, Vt., USA). FX antigen paired antibodies for ELISA wereobtained from Cedarlane (Burlington, Canada). RVV-X activator wasobtained from Diagnostica Stago (Paris, France), or HaematologicTechnologies. Restriction endonuclease Apal was obtained from NewEngland Biolabs (Ipswich, Mass., USA). T4-DNA ligase was obtained fromRoche (Roche Applied Science, Indianapolis, Ind., USA).

The DNA sequence encoding modified human FX-A is provided as SEQ IDNO:7. The DNA sequence encoding modified human FX-B is provided as SEQID NO:8. Nucleotides encoding SEQ ID NO:4 (to generate modified humanFX-A) or SEQ ID NO:5 (to generate modified human FX-B) sequences flankedby Apal restriction sites were synthesized by Genscript (Piscataway,N.J., USA), subcloned into pCMV4 mammalian expression vector using Apaland T4-DNA ligase and sequenced for consistency. Modified human FX-A andmodified human FX-B are also referred to as mod-hFX-A and mod-hFX-B,respectively. Stable HEK293 cell lines expressing r-hFX or modified hFXwere obtained as described previously (Larson et al., Biochemistry 1998,37:5029-5038). HEK293 cells were cotransfected with pCMV4 and pcDNA-PACEvectors using Lipofectamine2000 according to the manufacturer'sinstructions. FX expression of transfectants was assessed by a modifiedone-step clotting assay using FX-depleted human plasma. Transfectantswith the highest expression levels were expanded into T175 cultureflasks and conditioned for 24 hours on expression media (DMEM-F12nutrient mixture without Phenol-red supplemented with:Penicillin/Streptomycin/Fungizone, 2 mM L-glutamine, 10 μg/ml ITS, 100μg/ml Geneticin-418 sulphate and 6 μg/ml vitamine K). Conditioned mediawas collected, centrifuged at 10,000 g to remove cellular debris,concentrated in a 10-kDa cut-off filter (Millipore, Darmstadt, Germany),washed with HEPES-buffered saline and stored in 50% glycerol at −20° C.FX antigen levels of glycerol stocks were assessed by sandwich ELISAaccording to the manufacturer's instructions using human pooled plasmaas reference, assuming a plasma FX concentration of 10 μg/ml.

Expression media was conditioned for 24 hours on stable cell linesexpressing either r-hFX, modified human FX-A or modified human FX-B. Analiquot of conditioned media was incubated with RVV-X (10 ng/l;Haematologic Technologies) for 120 minutes at 37° C. After activation,modified human FX-A or modified human FX-B are also referred to asm-hFXa A or m-hFXa B, respectively. Assuming similar substrateaffinities for all FXa variants, the concentration of FXa in media wassubsequently determined by peptidyl substrate conversion (Spec-Xa, 250μM) using known concentrations of pd-hFXa as reference. Steady-stateinitial velocities of macromolecular substrate cleavage were determineddiscontinuously at 25° C. as described (Camire, J. Biol. Chem. 2002,277:37863-70). Briefly, progress curves of prothrombin activation wereobtained by incubating PCPS (50 μM), DAPA (10 μM), and prothrombin (1.4μM) with human recombinant FV-810 (B-domain truncated, constitutivelyactive), and the reaction was initiated with either 0.1 nM of pd-hFXa,r-hFXa, m-hFXa B, or 0.033 nM of m-hFXa A. The rate of prothrombinconversion was measured as described (Krishnaswamy et al., Biochemistry1997, 36:3319-3330).

Recombinant FX and modified human FX-A and modified human FX-B (200 ng)were activated by RVV-X (0.5 U/ml) for 60 minutes at 37° C. andsubjected to electrophoresis under reducing (30 mM dithiothreitol)conditions using pre-cast 4-12% gradient gels and the MES buffer system(Life Technologies) and transferred to a nitrocellulose membrane usingthe Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Hercules,Calif., USA). The blot was probed with an anti-heavy chain FX antibodyand protein bands were visualized using a Dyelight-800 anti-mousefluorescent antibody (Thermo Scientific, Rockford, Ill. USA).Plasma-derived hFXa (200 ng) was used as a reference.

Thrombin generation was adapted from protocols earlier described (Hemkeret al., Pathophysiol. Haemost. Thromb. 2003, 33:4-15). Briefly,FX-depleted plasma was mixed with Corn Trypsin Inhibitor (70 μg/ml),buffer (25 mM HEPES, 175 mM NaCl, 5 mg/ml BSA, pH 7.5) and PCPS (20 μM)and incubated for 10 minutes at 37° C. in a 96-well microplate. Thrombinformation was initiated by addition of pd-hFXa (0.5 nM) or vpt-FXa (0.5nM) preincubated with Rivaroxaban (0.4 μM) or Apixaban (0.2 μM),supplemented with FluCa and immediately transferred to the plasma mix.The final reaction volume was 120 μl, of which 64 μl was FX-depletedplasma. Thrombin formation was determined every 20 seconds for 30minutes and corrected for the calibrator using a software suite(Thrombinoscope, version 5.0). The mean endogenous thrombin potential(the area under the thrombin generation curve) was calculated from atleast two individual experiments. Calibrator and fluorescent substrate(FluCa) were purchased from Thrombinoscope (Maastricht, TheNetherlands).

Peptidyl substrate conversion (Spec-Xa, 250 μM final) of each FXavariant was performed in the absence or presence of direct FXainhibitors Rivaroxaban and Apixaban (0.001 μM-100 μM final) at ambienttemperature. Calcium-free stocks of pd-hFXa (2 nM final) or vpt-FXa (10nM final) were diluted in assay buffer and incubated in a 96-wellmicroplate in the presence of assay buffer or inhibitor for 2 minutes.Substrate conversion was initiated with Spec-Xa and absorption wasmonitored for 10 minutes at 405 nM in a SpectraMax M2e microplate readerequipped with the Softmax Pro software suite (Molecular Devices,Sunnyvale, Calif., USA). In order to assay DFXI sensitivity of eachrecombinant FX variant, glycerol stocks (5-40 μl) of r-hFX, modifiedhuman FX-A and modified human FX-B were diluted in assay buffer andincubated with RVV-X (0.5 U/ml) for 60 minutes at 37° C. Activatedstocks were subsequently diluted in assay-buffer, incubated for 2minutes in a 96-well microplate in the presence of assay buffer orinhibitor and assayed for substrate conversion as described. Therelative concentration of rhFX, m-hFXa A and m-hFXa B was assessed fromthe rate of substrate conversion in the absence of inhibitor using knownconcentrations of pd-hFXa as reference.

Results

Venom-Derived P. textilis (Vpt)-FXa is Resistant to Inhibition by DFXIs

Biochemical characterization of purified recombinant venom-derived P.textilis FXa (vptFXa) revealed that this protease, unlike any other FXaspecies known to date, is resistant to inhibition by the directanticoagulants rivaroxaban and apixaban, which have been designed toreversibly block the active site of FXa. Consistent with previousobservations, the Ki for human FXa (hFXa) inhibition was approximately 1nM (Perzborn, J. Thromb. Haemost. 2005, 3:514-521), whereas vptFXainhibition was at least a 1000-fold reduced (FIG. 2A). These findingswere corroborated in a plasma system mimicking in vivo fibringeneration, demonstrating that physiological concentrations of the FXainhibitors hardly affected vptFXa-initiated thrombin formation, while asignificant reduction was observed with hFXa present (FIGS. 2B and 2C).

Human-Venom P. textilis FXa Chimeras

A striking structural element that is not only limited to vptFXa, butalso present in venom FX from the Australian snake Notechis scutatus, isan altered amino acid composition at a position close to the hFXa activesite (FIG. 1, Panel C). Given its location, it was hypothesized thatthis unique helix may not only modulate the interaction with rivaroxabanand/or apixaban, but also with FVa, as the FVa binding site isC-terminal to this helix (Lee et al., J. Thromb. Haemost. 2011,9:2123-2126). To test this hypothesis, the two-protein coding DNAconstructs as listed in SEQ ID NOS:7 and 8 were prepared. The mod-hFX-Achimera as provided in SEQ ID NO:7 comprises the relevant part of the N.scutatus DNA sequence (indicated in bold and underlined) and themod-hFX-B chimera as provided in SEQ ID NO:8 comprises the relevant partof the P. textilis sequence (indicated in bold and underlined).

Using these DNA constructs, HEK293 cell lines were generated that stablyproduced both chimeric proteins and subsequently assessed the expressionlevels of modified human FX from HEK293 cells by conditioning the cellson expression media for 24 hours. Western blot analysis revealedexpression of full-length FX for both chimeric variants similar towild-type FX (FIG. 3A). Incubation with activator from Russell's ViperVenom (RVV-X) resulted in proteolytic activation of approximately 30% ofzymogen FX to FXa, indicated by the appearance of the ˜29 kDa heavychain band. The heavy chain of both modified human FXa-A and modifiedhuman FXa-B migrated at a slightly higher molecular weight, which isconsistent with the insertion of a snake sequence that is 12 or 13residues longer as compared to that of human FXa, respectively. Analysisof the FX antigen levels in conditioned media indicated that whereas theexpression of mod-hFX-A was approximately seven-fold reduced, that ofmod-hFX-B was similar to wild-type human FX (FIG. 3B). The low FXantigen levels of mod-hFX-A correlated with the similarly low FXactivity levels observed employing a modified clotting assay. Thisindicates that while the protein expression of mod-hFX-A is suboptimalas compared to that of the other FX variants, its FX function is notperturbed.

To test zymogen activation of FX, rFX and modified human FX-A andmodified human FX-B was converted to FXa using FX activator fromRussell's Viper Venom (RVV-X). Both modified human FXa-A and modifiedhuman FXa-B displayed protease activity upon RVV-X activation, asassessed by conversion of the small FXa-specific peptidyl substrateSpectroZyme Xa. In addition, the prothrombin conversion rates in thepresence of the human cofactor FVa of both chimeras were similar tohuman FXa (both pd-hFXa and r-hFXa) (FIG. 4). Collectively, theseobservations suggest that the snake sequence insertions do not severelyhamper the enzymatic properties of human FX.

Inhibition of FXa Chimeras by DFXIs

To estimate the inhibitory constant (Ki) of Rivaroxaban and Apixaban forRVV-X activated modified human FX-A, the activated recombinant proteinwas pre-incubated with 0.001 to 100 μM of inhibitor and subsequentlyassayed for its catalytic activity toward SpectroZyme Xa. Whileincubation with 0.5 μM Rivaroxaban resulted in full inhibition of r-hFXaand pd-hFXa, mod-hFXa-A remained fully active under these conditions(FIG. 5A). Moreover, the chimeric variant still displayed partialchromogenic activity following incubation with 100 μM Rivaroxaban,similar to the P. textilis venom FXa. These data indicate that the Kifor inhibition of mod-hFXa-A is at least 100-fold increased as comparedto that of human FXa. A similar reduced sensitivity for inhibition byApixaban was observed (FIG. 5B).

Assessment of the inhibition of mod-hFXa-B by rivaroxaban and apixabanresulted in a Ki similar to that observed for mod-hFXa-A (FIGS. 6A and6B). Thus, a reduced sensitivity for inhibition by apixaban andrivaroxaban was shown. Finally, DFXI-inhibition of the chimeric FXavariants was not altered in the presence of the cofactor FVa andnegatively charged phospholipid vesicles, suggesting that both the freeprotease as well as that assembled into an FVa-FXa-lipid-bound complexare equally resistant to inhibition by Rivaroxaban and Apixaban (FIGS.7A and 7B).

Example 2

Materials and Methods

Unless indicated otherwise, materials and methods as used in thisexample were the same or similar to the materials and methods indicatedin Example 1.

Construction and expression of recombinant FX: DNA encoding chimericFX-A (c-FX A), chimeric FX-B (c-FX B) and chimeric FX-C (c-FX C) weresynthesized at Genscript (Piscataway, N.J., USA), subcloned into pCMV4mammalian expression vector using Apal and T4-DNA ligase and sequencedfor consistency. Stabile HEK293 cell lines expressing recombinant humanor recombinant chimeric FX were obtained as described previously (Larsonet al., Biochemistry 1998, 37:5029-5038). HEK293 cells werecotransfected with pCMV4 and pcDNA-PACE vectors by LIPOFECTAMINE® 2000according to the manufacturer's instructions.

Purification of chimeric FX(a): Recombinant chimerix FX products A, Band C were prepared, purified and characterized as described previously(Camire et al., 2000), with the exception that the immunoaffinitypurification was replaced by a calcium gradient purification of FX on aPOROS HQ20-sepharose column. The typical yield of fully γ-carboxylatedrecombinant FX was 0.9 mg/liter conditioned medium. Purified recombinantchimeric FX was activated with RVV-X (0.1 U/mg FX), isolated bysize-exclusion chromatography on a Sephacryl S200 HR column (Vt 460 ml)and stored at −20° C. in HBS containing 50% vol/vol glycerol. Purifiedproducts were visualized by Coomassie staining.

Macromolecular substrate activation: Steady-state initial velocities ofmacromolecular substrate cleavage were determined discontinuously at 25°C. as described (Camire, 2002). Briefly, progress curves of prothrombinactivation were obtained by incubating PCPS (50 μM), DAPA (10 μM), andprothrombin (1.4 μM) with human recombinant FV-810 (20 nM, B-domaintruncated, constitutively active FV), and the reaction was initiatedwith either 0.1 nM of pd-hFXa, r-hFXa, c-FXa A, c-FXa B or c-FXa C. Therate of prothrombin conversion was measured as described (Krishnaswamyet al., 1997). Prothrombin conversion was assayed in absence or presenceof direct FXa inhibitors Edoxaban (CAS Registry Number 912273-65-5;manufactured by Daiichi Sankyo, marketed as Savaysa) and Apixaban (0.001μM-100 μM final) in order to determine DOAC sensitivity of eachrecombinant FXa variant.

Thrombin generation assays: Thrombin generation was adapted fromprotocols earlier described (Hemker et al., 2003). Briefly, thrombingeneration curves were obtained by supplementing FX-depleted plasma withTissue Factor (TF, 2 or 20 pM final), Corn Trypsin Inhibitor (70 μg/ml),PCPS (20 μM) and 1 Unit (prothrombin time-specific clotting activity) ofr-hFX (7 μg/ml) or chimeric FX-C (16 μg/ml). Thrombin formation wasinitiated by adding Substrate buffer (Fluca) to the plasma. FXa thrombingeneration curves were obtained by supplementing FX-depleted plasma withCorn Trypsin Inhibitor (70 μg/ml), assay buffer and PCPS (20 μM).Thrombin formation was initiated by addition of FXa premixed withRivaroxaban or Apixaban, assay buffer without calcium and supplementedwith Fluca. The final reaction volume was 120 μl, of which 64 μl wasFX-depleted plasma. Thrombin formation was determined every 20 secondsfor 30 minutes and corrected for the calibrator, using the software ofThrombinoscope. The lag time, mean endogenous thrombin potential (thearea under the thrombin generation curve), time to peak and peakthrombin generation, was calculated from at least three individualexperiments.

Results

The 9-13 residue insertion in the serine protease domains of P. textilisvenom, P. textilis isoform and N. scutatis venom FXa has promptedconstruction of chimeras of human and snake FX. Three protein coding DNAconstructs were made that incorporate each of these insertions in humanFXa (FIG. 9). Using these DNA constructs, HEK293 cell lines weregenerated that stably produce either recombinant normal human FX (r-hFX)or three types of chimeric FX (c-FX A, c-FX B and c-FX C). Expressionlevels of recombinant human and chimeric FX from HEK293 cells weredetermined by culturing the cells on expression media for 24 hours afterwhich the clotting activity of conditioned medium was assessed by amodified one-step PT clotting assay in FX-depleted plasma. Recombinantγ-carboxylated FX was purified from conditioned media by successiveion-exchange chromatography steps. A fraction of the FX pool wassubsequently activated with the FX activator from Russell's Viper venom,isolated by size-exclusion chromatography and characterized by SDS-PAGE.The heavy chain of purified plasma-derived factor Xa migrates as a 50/50mixture of FXa-α and FXa-β at ˜34-31 kDa. While autoproteolytic excisionof the C-terminal portion of FXa-α (residues 436-447) yields the β formof FXa, both isoforms are functionally similar with respect toprothrombinase assembly, prothrombin activation, antithrombinrecognition, and peptidyl substrate conversion (Pryzdial and Kessler,1996).

The purified products of r-hFXa and chimeric FXa-B and -C migratedpredominantly as FXa-β, chimeric FXa-A migrates as a 50/50 mixture of αand β FXa instead (FIG. 10, Panel A). Kinectis of macromolecularsubstrate activation by r-hFXa and chimeric FXa (A/B/C) on negativelycharged phospholipid vesicles (PCPS) in the presence of the cofactor FVashows that all chimeric variants assemble into the prothrombinasecomplex. However, the catalytic rate of chimeric FXa variants -A, -B and-C is respectively 8.2-, 6.8-, and 2.3-fold reduced compared torecombinant human FXa. Furthermore, recombinantly prepared human FXashows a modest decrease in catalytic efficiency compared toplasma-derived FXa (FIG. 10, Panel B).

To determine the inhibitory constant (Ki) of DOACs (Apixaban, Edoxaban)for chimeric FXa (A/B/C), the kinetics of prothrombin activation in thepresence of 0.001 to 100 μM of DOAC was assayed. While plasma-derivedFXa and recombinant human FXa are fully inhibited at near equimolarconcentrations of DOAC, all chimeric FXa variants were able to sustainprothrombin conversion at significantly higher FXa-inhibitorconcentrations (Ki Apixaban: 130-1270 nM, Ki Edoxaban: 3-270 nM) (FIG.11). Given that the chimeric FXa variants comprise similarly positionedinsertions with a varying length and composition of amino acids, theclose proximity of these insertions to the DOAC-coordinating residueTyr99 and/or the active site was speculated to be of direct consequenceto the decreased sensitivity for the DOACs.

In order to assess the potential of chimeric FXa to restore thrombingeneration in DOAC-spiked plasma, a thrombin generation (TG) assay wasperformed. FXa-initiated (5 nM) thrombin generation in FX-depleted humanplasma demonstrated a normal TG profile for c-FXa variant C, and nearnormal profiles for c-FXa variants A and B (FIG. 12A). While Apixaban (2μM) dramatically prolonged the lag time and reduced peak thrombingeneration in pd-FXa- and r-hFXa-initiated TG, these parameters wereunperturbed with the chimeric FXa variants present (FIG. 12B) (Table 2).These results show that chimeric FXa variants are able to restorehemostasis in DOAC-inhibited plasma. In addition, the zymogen form ofchimeric FX-C is also able to sustain thrombin generation in FX-depletedplasma. Initiation of coagulation by a low Tissue Factor (TF, 2 μM)concentration generates a robust TG curve for chimeric FX-C that is notaffected by Apixaban, unlike TG by r-hFX (FIG. 13A). At low TFconcentration, chimeric FX-C displays a short delay in the onset of TGand time to peak; in addition, chimeric FX-C has a larger endogenousthrombin potential (ETP) and higher peak thrombin generation (Table 3).However, these values normalize at high TF (20 μM) concentrations (FIG.13B) (Table 3). Based on the observations made in the FXa-initiated TGassay, it is expected that zymogen forms of chimeric FX variants A and Balso sustain TF-initiated TG in DOAC-spiked plasma. FIGS. 14A and 14B,in combination with Table 4, provide further evidence the effect ofchimeric FXa variants on restoring hemostasis in DOAC-inhibited plasma.Taken together, these results show that chimeric FX(a) is able torestore hemostasis in DOAC-inhibited plasma, both in zymogen andprotease form.

TABLE 2 Effect of Apixaban on FXa-initiated TG parameters. Valuesrepresent experimental TG values obtained in the presence of Apixabancorrected for TG values obtained inthe absence of Apixaban. pd-FXar-hFXa c-FXa -A c-FXa -B c-FXa -C Lagtime arrest 299 293 61 12 3(seconds) Delay in time to 515 467 120 30 7 peak (seconds) Peak Thrombin26 33 78 85 99 Generation (% of no Apixaban) Area under the 67 76 89 92101 curve (% of no Apixaban)

TABLE 3 Summary of low and high TF-initiated TG experiments. r-hFX +c-FX -C + r-hFX + c-FX -C + Low TF (2 pM) r-hFX Apixaban c-FX -CApixaban High TF (20 pM) r-hFX Apixaban c-FX -C Apixaban Lagtime 132 ±5  696 ± 162 185 ± 12 186 ± 6  Lagtime 48 ± 1 138 ± 12  72 ± 2  78 ± 2(seconds) (seconds) Time to peak 324 ± 6  no peak 480 ± 24 492 ± 23 Timeto peak 114 ± 6  804 ± 36 138 ± 6 144 ± 9 (seconds) (seconds) Peakthrombin 61 ± 4 8 ± 4 78 ± 1 72 ± 4 Peak thrombin 338 ± 8  32 ± 4  334 ±15  321 ± 15 (nM) (nM) ETP (nM) 567 ± 61 no ETP  830 ± 131 756 ± 38 ETP(nM) 973 ± 18 694 ± 67 1027 ± 19 1012 ± 33

TABLE 4 Effect of Edoxaban on TF-initiated TG parameters for r-hFX andc-FX-C. Values represent experimental TG values obtained in the presenceof increasing concentrations of Edoxaban. Edoxaban (nM) control 50 100200 400 600 1000 2000 r-hFX Lagtime(s) 115 247 297 397 538 679 874 1180ETP remaining 100.% 99.3 87.1 no ETP no ETP no ETP no ETP no ETP Peakheight % 100.% 41.2 30.8 23.1 15.9 13.0 10.1 7.1 Time to peak(s) 265 618756 1290 1890 1932 2472 2562 c-FX -C Lagtime(s) 188 161 161 172 182 197212 232 ETP remaining 100.% 87.9 92.5 88.3 92.7 96.8 92.7 82.0 Peakheight % 100.% 109.3 112.3 101.0 94.6 88.7 77.5 65.3 Time to peak(s) 433382 388 418 448 483 538 578

What is claimed is:
 1. A recombinant protein comprising a coagulation factor Xa polypeptide, said coagulation factor Xa polypeptide having an alteration or deletion of an amino acid residue corresponding to amino acid residue Phe-396 as indicated in SEQ ID NO:
 1. 2. The protein according to claim 1, wherein the alteration or deletion of an amino acid residue corresponding to amino acid residue Phe-396 is combined with an insertion of 1-50 amino acid residue(s) in a region corresponding to the region of amino acid residues between Gly-289 and Asp-320.
 3. The protein according to claim 1, wherein the alteration or deletion of an amino acid residue corresponding to amino acid residue Phe-396 is combined with an insertion of 1-50 amino acid residue(s) in a region corresponding to the region of amino acid residues between His-311 and Asp-320 of SEQ ID NO:
 1. 4. The protein according to claim 3, wherein the region of amino acid residues corresponding to amino acid residues between His-311 and Asp-320 of SEQ ID NO: 1 has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:
 11. 5. A pharmaceutical composition comprising the protein of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 6. A method of completely or partially reversing an anti-coagulant effect of a direct factor Xa inhibitor in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of the protein of claim 1 so as to completely or partially reverse the anti-coagulant effect of the direct factor Xa inhibitor in the subject.
 7. The method according to claim 6, wherein the direct factor Xa inhibitor is rivaroxaban (5-chloro-N-[[(5S)-2-oxo-3-[4-(3-oxo-4-morpholinyl)phenyl]-5-oxazolidinyl]methyl]-2-thiophenecarboxamide), apixaban (1-(4methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide), edoxaban (N′-(5-chloropyridin-2-yl)-N-[(1S,2R,4S)-4-(dimethylcarbamoyl)-2-[(5-methyl-6,7-dihydro-4H-[1,3]thiazolo[5,4-c]pyridine-2-carbonyl)amino]cyclohexyl]oxamide; 4-methylbenzenesulfonic acid), or betrixaban (N-(5-chloropyridin-2-yl)-2-[[4-(N,N-dimethylcarbamimidoyl)benzoyl]amino]-5-methoxybenzamide).
 8. A method of making the protein of claim 1, the method comprising: expressing a nucleic acid molecule comprising a DNA sequence that encodes the protein in a host cell.
 9. The method according to claim 8, wherein the nucleic acid molecule is comprised within an expression vector. 